The genome and proteome of the Kluyvera bacteriophage Kvp1 – another member of the T7-like Autographivirinae
© Lingohr et al; licensee BioMed Central Ltd. 2008
Received: 18 September 2008
Accepted: 20 October 2008
Published: 20 October 2008
Kluyvera, a genus within the family Enterobacteriaceae, is an infrequent cause of human infections. Bacteriophage Kvp1, the only bacteriophage isolated for one of its species, Kluyvera cryocrescens, is a member of the viral family Podoviridae.
The genome of Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was sequenced using pyrosequencing (454 technology) at the McGill University and Genome Québec Innovation Centre. The two contigs were closed using PCR and the sequence of the terminal repeats completed by primer walking off the phage DNA. The phage structural proteome was investigated by SDS-PAGE and mass spectrometry.
At 39,472 bp, the annotated genome revealed a closer relationship to coliphage T3 than T7 with Kvp1 containing homologs to T3 early proteins S-adenosyl-L-methionine hydrolase (0.3) and protein kinase (0.7). The quantitative nature of the relationships between Kvp1 and the other members of the T7-like virus genus (T7, T3, φA1122, φYeO3-12, Berlin, K1F, VP4 and gh-1) was confirmed using CoreGenes.
The T7-like bacterial viruses are members of the Podoviridae – phages with short tails – and are characterized by a simple but elegant temporal transcriptional control system . The early genes are transcribed by the host RNA polymerase while the middle and late regions are transcribed by a single subunit phage-encoded RNA polymerase which recognizes unique 23 bp promoters sequences . These viruses are one of the most common types of bacteriophages with 26–29 defined or tentative species according to the VIII report of the International Committee on the Taxonomy of Viruses [3, 4]. Most of the host species are members of the γ-Proteobacteria (Erwinia, Escherichia, Klebsiella, Morganella, Pseudomonas, Salmonella, Vibrio, and Yersinia) but viral isolates also infecting α-Proteobacteria (Caulobacter, and Rhizobium) have been isolated. Fifteen T7-like phages have been sequenced and deposited with GenBank. As a result of a reanalysis, at the protein level, of relationships within the "T7-like viruses" this group of bacteriophages have been classified into the subfamily Autographivirinae which currently possesses three genera: T7-like, Sp6-like and φKMV-like viruses . Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was isolated from the Matanza River in Buenos Aires (Argentina) by Gadaleta and Zorzopulos . Morphologically this phage is a member of the Podoviridae. Eleven clones derived from AluI or HaeIII digestion of the viral DNA were sequenced, by these authors, revealing strong sequence similarity to coliphage T7. To further analyze the correct taxonomic position of this virus we have completed the sequence of its genome noting its very close similarity to Yersinia phage Berlin and coliphage T3.
Results and discussion
Pyrosequencing (454 technology) has been used to determine the sequence of the genomes of Bacillus thuringiensis phage 0305φ8-36  and coliphage JK98 , and, in this incidence, the genome of Kvp1. Sequencing resulted in 2 contigs with 53-fold coverage. While this type of sequencing can result in potential errors at oligonucleotide runs, none were observed in the data on Kvp1. The gap, representing 0 bp, was closed by PCR amplification and ABI sequencing; while the nature of the termini were verified by primer walking off phage DNA template. The total genome is 39,472 bp with 194 bp terminal direct repeats, and a base composition of 48.6 mol%G+C – characteristics remarkably consistent with other T7-like phages. By comparison, the genomes of T7-like phages range from 37.4 kb (Pseudomonas phage gh-1) to 45.4 kb (Erwinia phage Era103) while the reported terminal repeats range from Yersinia phage φA1122 at 148 bp to Pseudomonas aeruginosa phage LKD16 at 428 bp.
No tRNA genes were discovered, which was not an unexpected observation since no T7-like phages have been found to harbour them; 46 ORFs were delineated encoding protein products which show the strongest sequence similarity to gene products (Gps) from Yersinia phage Berlin (NC_008694). To investigate the relationships further we employed two homology tools, one of which function at the DNA sequence level (Mauve) and one, CoreGenes, which compares proteins.
Our data conclusively demonstrate that Kluyvera virus Kvp1 is a member of T7-like virus genus of the Podoviridae subfamily Autographivirinae. It differs from phages such as T3 and φYeO3-12 which exhibit capsid frameshifting at lysyl residues, by ribosomal slippage at polyU residues (phenylalanine) – a property it shares with Yersinia phage φA1122.
Materials and methods
Purification of phage wV8
Bacteriophage Kvp1 (HER400) and its host K. cryocrescens strain HER1400 were received from the Felix d'Hérelle Reference Center for Bacterial Viruses at Université Laval (Québec, QC, Canada). The phage was propagated at 30°C using standard protocols, precipitated using polyethylene glycol 8000 and purified through two rounds of CsCl equilibrium gradient centrifugation .
The DNA was isolated using the SDS-proteinase K protocol of Sambrook and Russell (2001) and was submitted to the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) for DNA sequencing. This resulted in two contigs which were closed using PCR with custom primers and, standard dideoxy sequencing of the amplicon (University of Guelph, Laboratory Services, Guelph, ON, Canada). The termini were determined by primer walking.
The genome was screened for tRNA-encoding genes using Aragorn  and tRNAScan ; and, for protein encoding genes using Kodon (Applied Maths, Austin, TX) and PSI-BLAST . Rho-independent terminators identified using TransTerm  at http://nbc11.biologie.uni-kl.de/framed/left/menu/auto/right/clusterinfo2. Phage-specific promoters were discovered using PHIRE  and displayed using WebLogo . The sequence of this bacteriophage has been deposited with GenBank (accession no. FJ194439).
Whole genome comparisons
SDS-PAGE  was carried out on CsCl-purified phage particles along with the PageRuler Unstained Protein Ladder (Fermentas, Burlington, ON, Canada) stained with Coomassie brilliant blue R250 and characterized using Bionumerics software (Applied Maths). Bands were further characterized by in situ trypsin digestion and mass spectrometry. Briefly, the excised gel bands were destained until colorless, and dried using a SpeedVac. Following reduction with DTT and alkylation with iodoacetamide, the protein was digested with 10 ng of sequencing grade trypsin (Calbiochem) in 25 mM NH4HCO3 (pH 7.6) at 37°C overnight. The proteolytic peptides were extracted, and cleaned up by a C18 Ziptip (Millipore). MALDI data were acquired using an Applied Biosystems/MDS Sciex QStar XL quadrupole time-of-flight (QqTOF) mass spectrometer under a nitrogen laser (337 nm), and 2,5-dihydroxybenzoic acid was used as the matrix. All peptide fingerprinting masses (m/z) on the MS spectrum were compared with the theoretical values generated in-silico by MS-Digest, a ProteinProspector program developed in the UCSF Mass Spectrometry Facility http://prospector.ucsf.edu/. The individual peptide sequence was identified by MALDI MS/MS measurements on the same instrument using argon as the collision gas.
matrix-assisted laser desorption ionization
- QqTOF MS:
quadrupole time-of-flight mass spectrometry
tandem mass spectrometry.
A.K. is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. We thank Rob Lavigne for his critical review of the MS. P-J.C. holds a predoctoral fellowship from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (I.W.T., Belgium).
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